Search Results (25)

Domain-specific O-fucosylation is an unusual type of glycosylation, where the fucose is directly attached to the serine or threonine residues in specific protein domains via an O-linkage. O-fucosylated proteins play critical roles in a wide variety of biological events and hold important therapeutic values, with the most studied being the Notch receptors and ADAMTS proteins. O-fucose glycans modulate the function of the proteins they modify and are closely associated with various diseases including cancer. In mammals, alongside the well-documented protein O-fucosyltransferase (POFUT) 1-mediated O-fucosylation of epidermal growth factor-like (EGF) repeats and POFUT2-mediated O-fucosylation of thrombospondin type 1 repeats (TSRs), a new type of O-fucosylation was recently identified on elastin microfibril interface (EMI) domains, mediated by POFUT3 and POFUT4 (formerly FUT10 and FUT11). In this review, we present an overview of our current knowledge of O-fucosylation, integrating the latest findings and with a particular focus on its biological functions and molecular mechanisms. Full article

Basilepta melanopus is a pest that severely affects oil tea plants, and the Notch signaling pathway plays a significant role in the early development of insect ovaries. In this study, we explored the function of the notch gene within the Notch signaling pathway in the reproductive system of B. melanopus. The functional domains and expression patterns of Bmnotch were analyzed. Bmnotch contains 45 epidermal growth factor-like (EGF-like) domains, one negative regulatory region, one NODP domain and one repeat-containing domain superfamily. The qPCR reveals heightened expression in early developmental stages and specific tissues like the head and ovaries. The RNA interference (RNAi)-based suppression of notch decreased its expression by 52.1%, exhibiting heightened sensitivity to dsNotch at lower concentrations. Phenotypic and mating experiments have demonstrated that dsNotch significantly impairs ovarian development, leading to reduced mating frequencies and egg production. This decline underscores the Notch pathway’s crucial role in fecundity. The findings advocate for RNAi-based, Notch-targeted pest control as an effective and sustainable strategy for managing B. melanopus populations, signifying a significant advancement in forest pest control endeavors. Full article

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His₆-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation. Full article

Notch signaling is conserved in C. elegans, Drosophila, and mammals. Among the four NOTCH genes in humans, NOTCH1, NOTCH2, and NOTCH3 are known to cause monogenic hereditary disorders. Most NOTCH-related disorders are congenital and caused by a gain or loss of Notch signaling activity. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 is adult-onset and considered to be caused by accumulation of the mutant NOTCH3 extracellular domain (N3ECD) and, possibly, by an impairment in Notch signaling. Pathophysiological processes following mutant N3ECD accumulation have been intensively investigated; however, the process leading to N3ECD accumulation and its association with canonical NOTCH3 signaling remain unknown. We reviewed the progress in clarifying the pathophysiological process involving mutant NOTCH3. Full article

Aerobic exercise, specifically high-intensity interval exercise (HIIE), and its effects on renal health and filtration (RHF) are not well understood. Several studies support incorporating contemporary biomarkers serum cystatin C (CyC) and urine epidermal growth factor (uEGF) to combat the volatility of serum creatinine (sCr). Using these biomarkers, we examined the acute influences HIIE has on RHF to determine if there is a ceiling effect in healthy populations. The purpose was to determine the influence of an acute bout of HIIE on RHF. Thirty-six participants (n = 22 males; n = 14 females; age 37.6 ± 12.4 years.; BF% 19.2 ± 7.1%; VO₂max 41.8 + 7.4 mL/kg/min) completed 30 min of HIIE on a treadmill (80% and 40% of VO₂reserve in 3:2 min ratio). Blood and urine samples were obtained under standardized conditions before, 1 h, and 24 h post-exercise. CyC, sCR, uEGF, urine creatinine (uCr), uCr/uEGF ratio, and multiple estimates of glomerular filtration rate (eGFR) Modification of Diet in Renal Disease (MDRD) and CKD-EPI equations were used. The analysis employed paired sample t-tests and repeated measures ANOVAs. CyC, uEGF, uCr, and uCr/uEGF ratio concentrations were not altered between timepoints. sCr increased 1 h post-exercise (p > 0.002) but not at 24 h post-exercise. eGFR decreased in the MDRD and CKD-EPI equations at 1 h (p > 0.012) with no changes at 24 h post-exercise. CyC and sCr/CyC demonstrated no significant changes. CyC and uEGF are not altered by acute HIIE. The results demonstrate a potential ceiling effect in contemporary and traditional biomarkers of RHF, indicating improvements in RHF may be isolated to populations with reduced kidney function. Full article

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma. Full article

Notch signaling plays various roles in cell-fate specification through direct cell–cell interactions. Notch receptors are evolutionarily conserved transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats. Drosophila Notch has 36 EGF-like repeats, and while some play a role in Notch signaling, the specific functions of most remain unclear. To investigate the role of each EGF-like repeat, we used 19 previously identified missense mutations of Notch with unique amino acid substitutions in various EGF-like repeats and a transmembrane domain; 17 of these were identified through a single genetic screen. We assessed these mutants’ phenotypes in the nervous system and hindgut during embryogenesis, and found that 10 of the 19 Notch mutants had defects in both lateral inhibition and inductive Notch signaling, showing context dependency. Of these 10 mutants, six accumulated Notch in the endoplasmic reticulum (ER), and these six were located in EGF-like repeats 8–10 or 25. Mutations with cysteine substitutions were not always coupled with ER accumulation. This suggests that certain EGF-like repeats may be particularly susceptible to structural perturbation, resulting in a misfolded and inactive Notch product that accumulates in the ER. Thus, we propose that these EGF-like repeats may be integral to Notch folding. Full article

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation. Full article

The pathogenesis of multiple sclerosis (MS) remains enigmatic and controversial. Myelin sheaths in the central nervous system (CNS) insulate axons and allow saltatory nerve conduction. MS brings about the destruction of myelin sheaths and the myelin-producing oligodendrocytes (ODCs). The conundrum of remyelination failure is, therefore, crucial in MS. In this review, the roles of epidermal growth factor (EGF), normal prions, and cobalamin in CNS myelinogenesis are briefly summarized. Thereafter, some findings of other authors and ourselves on MS and MS-like models are recapitulated, because they have shown that: (a) EGF is significantly decreased in the CNS of living or deceased MS patients; (b) its repeated administration to mice in various MS-models prevents demyelination and inflammatory reaction; (c) as was the case for EGF, normal prion levels are decreased in the MS CNS, with a strong correspondence between liquid and tissue levels; and (d) MS cobalamin levels are increased in the cerebrospinal fluid, but decreased in the spinal cord. In fact, no remyelination can occur in MS if these molecules (essential for any form of CNS myelination) are lacking. Lastly, other non-immunological MS abnormalities are reviewed. Together, these results have led to a critical reassessment of MS pathogenesis, partly because EGF has little or no role in immunology. Full article

Notch signaling, which was initially identified in Drosophila wing morphogenesis, plays pivotal roles in cell development and differentiation. Optimal Notch pathway activity is essential for normal development and dysregulation of Notch signaling leads to various human diseases, including many types of cancers. In hematopoietic cancers, such as T-cell acute lymphoblastic leukemia, Notch plays an oncogenic role, while in acute myeloid leukemia, it has a tumor-suppressive role. In solid tumors, such as hepatocellular carcinoma and medulloblastoma, Notch may have either an oncogenic or tumor-suppressive role, depending on the context. Aberrant expression of Notch receptors or ligands can alter the ligand-dependent Notch signaling and changes in trafficking can lead to ligand-independent signaling. Defects in any of the two signaling pathways can lead to tumorigenesis and tumor progression. Strikingly, O-glycosylation is one such process that modulates ligand–receptor binding and trafficking. Three types of O-linked modifications on the extracellular epidermal growth factor-like (EGF) repeats of Notch receptors are observed, namely O-glucosylation, O-fucosylation, and O-N-acetylglucosamine (GlcNAc) modifications. In addition, O-GalNAc mucin-type O-glycosylation outside the EGF repeats also appears to occur in Notch receptors. In this review, we first briefly summarize the basics of Notch signaling, describe the latest information on O-glycosylation of Notch receptors classified on a structural basis, and finally describe the regulation of Notch signaling by O-glycosylation in cancer. Full article

Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG. Full article

Aryl hydrocarbon receptor (AHR) genomic pathway has been well-characterized in a number of respiratory diseases. In addition, the cytoplasmic AHR protein may act as an adaptor of E3 ubiquitin ligase. In this study, the physiological functions of AHR that regulate cell proliferation were explored using the CRISPR/Cas9 system. The doubling-time of the AHR-KO clones of A549 and BEAS-2B was observed to be prolonged. The attenuation of proliferation potential was strongly associated with either the induction of p27^Kip1 or the impairment in mitogenic signal transduction driven by the epidermal growth factor (EGF) and EGF receptor (EGFR). We found that the leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a repressor of EGFR, was induced in the absence of AHR in vitro and in vivo. The LRIG1 tends to degrade via a proteasome dependent manner by interacting with AHR in wild-type cells. Either LRIG1 or a disintegrin and metalloprotease 17 (ADAM17) were accumulated in AHR-defective cells, consequently accelerating the degradation of EGFR, and attenuating the response to mitogenic stimulation. We also affirmed low AHR but high LRIG1 levels in lung tissues of chronic obstructive pulmonary disease (COPD) patients. This might partially elucidate the sluggish tissue repairment and developing inflammation in COPD patients. Full article

Repeated mechanical stress causes injuries in the adult skeletal muscle that need to be repaired. Although muscle regeneration is a highly efficient process, it fails in some pathological conditions, compromising tissue functionality. This may be caused by aberrant cell–cell communication, resulting in the deposition of fibrotic and adipose infiltrates. Here, we investigate in vivo changes in the profile of skeletal muscle secretome during the regeneration process to suggest new targetable regulatory circuits whose failure may lead to tissue degeneration in pathological conditions. We describe the kinetic variation of expression levels of 76 secreted proteins during the regeneration process. In addition, we profile the gene expression of immune cells, endothelial cells, satellite cells, and fibro-adipogenic progenitors. This analysis allowed us to annotate each cell-type with the cytokines and receptors they have the potential to synthetize, thus making it possible to draw a cell–cell interaction map. We next selected 12 cytokines whose receptors are expressed in FAPs and tested their ability to modulate FAP adipogenesis and proliferation. We observed that IL1α and IL1β potently inhibit FAP adipogenesis, while EGF and BTC notably promote FAP proliferation. In addition, we characterized the cross-talk mediated by extracellular vesicles (EVs). We first monitored the modulation of muscle EV cargo during tissue regeneration. Using a single-vesicle flow cytometry approach, we observed that EVs differentially affect the uptake of RNA and proteins into their lumen. We also investigated the EV capability to interact with SCs and FAPs and to modulate their proliferation and differentiation. We conclude that both cytokines and EVs secreted during muscle regeneration have the potential to modulate adipogenic differentiation of FAPs. The results of our approach provide a system-wide picture of mechanisms that control cell fate during the regeneration process in the muscle niche. Full article

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the epidermal growth factor (EGF)-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraines, psychiatric disorders, recurrent strokes, and dementia. Omic technologies allow the massive study of different molecules for understanding diseases in a non-biased manner or even for discovering targets and their possible treatments. We analyzed the progress in understanding CADASIL that has been made possible by omics sciences. For this purpose, we included studies that focused on CADASIL and used omics techniques, searching bibliographic resources, such as PubMed. We excluded studies with other phenotypes, such as migraine or leukodystrophies. A total of 18 articles were reviewed. Due to the high prevalence of NOTCH3 mutations considered pathogenic to date in genomic repositories, one can ask whether all of them produce CADASIL, different degrees of the disease, or whether they are just a risk factor for small vessel disease. Besides, proteomics and transcriptomics studies found that the molecules that are significantly altered in CADASIL are mainly related to cell adhesion, the cytoskeleton or extracellular matrix components, misfolding control, autophagia, angiogenesis, or the transforming growth factor β (TGFβ) signaling pathway. The omics studies performed on CADASIL have been useful for understanding the biological mechanisms and could be key factors for finding potential drug targets. Full article

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6. Full article